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Novel oral poliovirus vaccine type 2 (nOPV2) has been engineered to improve the genetic stability of Sabin oral poliovirus vaccine (OPV) and reduce the emergence of circulating vaccine-derived polioviruses. This trial aimed to provide key safety and immunogenicity data required for nOPV2 licensure and WHO prequalification. This phase 3 trial recruited infants aged 18 to <52 weeks and young children aged 1 to <5 years in The Gambia. Infants randomly assigned to receive one or two doses of one of three lots of nOPV2 or one lot of bivalent OPV (bOPV). Young children were randomised to receive two doses of nOPV2 lot 1 or bOPV. The primary immunogenicity objective was to assess lot-to-lot equivalence of the three nOPV2 lots based on one-dose type 2 poliovirus neutralising antibody seroconversion rates in infants. Equivalence was declared if the 95% CI for the three pairwise rate differences was within the –10% to 10% equivalence margin. Tolerability and safety were assessed based on the rates of solicited adverse events to 7 days, unsolicited adverse events to 28 days, and serious adverse events to 3 months post-dose. Stool poliovirus excretion was examined. The trial was registered as PACTR202010705577776 and is completed. Between February and October, 2021, 2345 infants and 600 young children were vaccinated. 2272 (96·9%) were eligible for inclusion in the post-dose one per-protocol population. Seroconversion rates ranged from 48·9% to 49·2% across the three lots. The minimum lower bound of the 95% CIs for the pairwise differences in seroconversion rates between lots was –5·8%. The maximum upper bound was 5·4%. Equivalence was therefore shown. Of those seronegative at baseline, 143 (85·6%) of 167 (95% CI 79·4–90·6) infants and 54 (83·1%) of 65 (71·7–91·2) young children seroconverted over the two-dose nOPV2 schedule. The post-two-dose seroprotection rates, including participants who were both seronegative and seropositive at baseline, were 604 (92·9%) of 650 (95% CI 90·7–94·8) in infants and 276 (95·5%) of 289 (92·4–97·6) in young children. No safety concerns were identified. 7 days post-dose one, 78 (41·7%) of 187 (95% CI 34·6–49·1) infants were excreting the type 2 poliovirus. nOPV2 was immunogenic and safe in infants and young children in The Gambia. The data support the licensure and WHO prequalification of nOPV2. Bill & Melinda Gates Foundation.

Recently, genetically stable novel OPVs (nOPV) were developed by modifying the genomes of Sabin viruses of conventional OPVs to reduce the risk of reversion to neurovirulence and therefore the risk of generating circulating vaccine-derived polioviruses. There is a need for specific and sensitive methods for the identification and quantification of nOPV viruses individually and in mixtures for clinical trials and potentially for manufacturing quality control and environmental surveillance. In this communication, we evaluated and improved the quantitative multiplex one-step reverse transcriptase polymerase chain reaction (qmosRT-PCR) assay for the identification and quantification of nOPV viruses in samples with different formulations and virus concentrations and in virus-spiked stool samples. The assay was able to specifically identify at least 1 log10 CCID50/mL of each serotype in the presence of the two other serotypes at high concentrations (6–7 log10 CCID50/mL) in the same sample. In addition, the lowest viral concentration that the assay was able to detect in stool samples was 17 CCID50/mL for nOPV1 and nOPV2 viruses and 6 CCID50/mL for nOPV3. We also found high correlation between the expected and observed (by qmosRT-PCR) concentrations of spiked viruses in stool samples for all three nOPV viruses, with R-squared values above 0.95. The analysis of samples collected from an nOPV2 clinical trial showed that 100% of poliovirus type 2 was detected and few samples showed the presence of type 1 and 3 residuals from previous vaccinations with bOPV (at least 4 weeks prior vaccination with nOPV2), confirming the high sensitivity of the method. The qmosRT-PCR was specific and sensitive for the simultaneous identification and quantification of all three nOPV viruses. It can be used as an identity test during the nOPV manufacturing process and in evaluation of virus excretion in nOPV clinical trials.

Novel oral poliovirus vaccine type 2 (nOPV2) is being developed to reduce the rare occurrence of disease and outbreaks associated with the genetic instability of the Sabin vaccine strains. Children aged 1 to 5 years were enrolled in two related clinical studies to assess safety, immunogenicity, shedding rates and properties of the shed virus following vaccination with nOPV2 (two candidates) versus traditional Sabin OPV type 2 (mOPV2). The anticipated pattern of reversion and increased virulence was observed for shed Sabin-2 virus, as assessed using a mouse model of poliovirus neurovirulence. In contrast, there were significantly reduced odds of mouse paralysis for shed virus for both nOPV2 candidates when compared to shed Sabin-2 virus. Next-generation sequencing of shed viral genomes was consistent with and further supportive of the observed neurovirulence associated with shed Sabin-2 virus, as well as the reduced reversion to virulence of shed candidate viruses. While shed Sabin-2 showed anticipated A481G reversion in the primary attenuation site in domain V in the 5’ untranslated region to be associated with increased mouse paralysis, the stabilized domain V in the candidate viruses did not show polymorphisms consistent with reversion to neurovirulence. The available data from a key target age group for outbreak response confirm the superior genetic and phenotypic stability of shed nOPV2 strains compared to shed Sabin-2 and suggest that nOPV2 should be associated with less paralytic disease and potentially a lower risk of seeding new outbreaks.

Emergence of mutations is an inherent property of RNA viruses with several implications for their replication, pathogenesis, and evolutionary adaptation. Oral poliovirus vaccine (OPV), developed by Albert Sabin, is composed of live attenuated polioviruses of three serotypes that can revert to neurovirulence during replication in cell culture and in vaccine recipients. Recently, a new modified variant of Sabin 2 virus was developed by introducing changes in its genome, making it more genetically stable to prevent the reversion. The new strain was used to manufacture novel OPV2 (nOPV2), which was approved by the World Health Organization for emergency use to stop outbreaks caused by circulating vaccine-derived poliovirus (cVDPV2). Manufacture of this improved vaccine requires close attention to the genetic heterogenicity to ensure that the levels of the undesirable mutations are limited. Preliminary studies using whole-genome Illumina sequencing (NGS) identified several genomic sites where mutations tend to occur with regularity. They include VP1-I143T amino acid change at the secondary attenuation site; VP1-N171D, a substitution that modestly increases neurovirulence in mice; and VP1-E295K, which may reduce the immunogenicity of the nOPV2. Therefore, to ensure the molecular consistency of vaccine batches, the content of these mutants must be quantified and kept within specifications. To do this, we have developed quantitative, multiplex, one-step reverse-transcriptase polymerase chain reactions (qmosRT-PCRs) as simple methods for quantification of these mutations. Each method uses specific short TaqMan probes with different dyes for the analysis of both mutants and non-mutants in the same sample. The quantification is done using calibration curves developed using validated reference materials. To evaluate the sensitivity and the linearity of the qmosRT-PCR method, the mutant viruses were spiked in non-mutant viruses, and nOPV2 batches were used to validate the method. The spiked samples and the nOPV2 batches were analyzed by qmosRT-PCR and NGS assays. The results showed that qmosRT-PCR is sensitive enough to detect around 1% of mutants. The percentages of mutants determined by qmosRT-PCR correlate well with the results of the NGS. Further, the analysis of the nOPV2 batches showed that the results of qmosRT-PCR correlated well with the results of NGS. In conclusion, the qmosRT-PCR is a specific, sensitive, and linear method. It could be used for quality control of the nOPV2 batches.

To control circulating vaccine-derived type 2 poliovirus outbreaks, a more genetically stable novel Oral Poliovirus Vaccine type 2 (nOPV2) was developed by targeted modifications of Sabin 2 genome. Since the use of OPV2 made of Sabin 2 strain has been stopped, it is important to exclude the possibility that batches of nOPV2 are contaminated with Sabin 2 virus. Here, we report the development of a simple quantitative one-step reverse-transcription polymerase chain reaction assay for the detection and quantitation of Sabin 2 virus in the presence of overwhelming amounts of nOPV2 strain. The method is specific and linear within 8 log10 range even in the presence of relevant amounts of nOPV2 virus. It is sensitive, with a lower limit of detection of 0.2 CCID50/mL (an equivalent of 198 genome copies per mL), and generates reproducible results. This assay can be used for quality control and lot release of the nOPV2.

Sabin-strain oral polio vaccines (OPV) can, in rare instances, cause disease in recipients and susceptible contacts or evolve to become circulating vaccine-derived strains with the potential to cause outbreaks. Two novel type 2 OPV (nOPV2) candidates were designed to stabilize the genome against the rapid reversion that is observed following vaccination with Sabin OPV type 2 (mOPV2). Next-generation sequencing and a modified transgenic mouse neurovirulence test were applied to shed nOPV2 viruses from phase 1 and 2 studies and shed mOPV2 from a phase 4 study. The shed mOPV2 rapidly reverted in the primary attenuation site (domain V) and increased in virulence. In contrast, the shed nOPV2 viruses showed no evidence of reversion in domain V and limited or no increase in neurovirulence in mice. Based on these results and prior published data on safety, immunogenicity, and shedding, the nOPV2 viruses are promising alternatives to mOPV2 for outbreak responses.

AbstractTo address the evolving risk of circulating vaccine-derived poliovirus type 2 (cVDPV2), Global Polio Eradication Initiative (GPEI) partners are working closely with countries to deploy an additional innovative tool for outbreak response – novel oral polio vaccine type 2 (nOPV2). The World Health Organization’s (WHO) Prequalification program issued an Emergency Use Listing (EUL) recommendation for nOPV2 on 13 November 2020. The WHO’s EUL procedure was created to assess and list unlicensed vaccines, therapeutics and diagnostics to enable their use in response to a Public Health Emergency of International Concern (PHEIC). nOPV2 was the first vaccine to receive an EUL, paving the way for other emergency vaccines. In this report, we summarise the pathway for nOPV2 roll-out under EUL.

Use of oral live-attenuated polio vaccines (OPV), and injected inactivated polio vaccines (IPV) has almost achieved global eradication of wild polio viruses. To address the goals of achieving and maintaining global eradication and minimising the risk of outbreaks of vaccine-derived polioviruses, we tested novel monovalent oral type-2 poliovirus (OPV2) vaccine candidates that are genetically more stable than existing OPVs, with a lower risk of reversion to neurovirulence. Our study represents the first in-human testing of these two novel OPV2 candidates. We aimed to evaluate the safety and immunogenicity of these vaccines, the presence and extent of faecal shedding, and the neurovirulence of shed virus. In this double-blind, single-centre phase 1 trial, we isolated participants in a purpose-built containment facility at the University of Antwerp Hospital (Antwerp, Belgium), to minimise the risk of environmental release of the novel OPV2 candidates. Participants, who were recruited by local advertising, were adults (aged 18–50 years) in good health who had previously been vaccinated with IPV, and who would not have any contact with immunosuppressed or unvaccinated people for the duration of faecal shedding at the end of the study. The first participant randomly chose an envelope containing the name of a vaccine candidate, and this determined their allocation; the next 14 participants to be enrolled in the study were sequentially allocated to this group and received the same vaccine. The subsequent 15 participants enrolled after this group were allocated to receive the other vaccine. Participants and the study staff were masked to vaccine groups until the end of the study period. Participants each received a single dose of one vaccine candidate (candidate 1, S2/cre5/S15domV/rec1/hifi3; or candidate 2, S2/S15domV/CpG40), and they were monitored for adverse events, immune responses, and faecal shedding of the vaccine virus for 28 days. Shed virus isolates were tested for the genetic stability of attenuation. The primary outcomes were the incidence and type of serious and severe adverse events, the proportion of participants showing viral shedding in their stools, the time to cessation of viral shedding, the cell culture infective dose of shed virus in virus-positive stools, and a combined index of the prevalence, duration, and quantity of viral shedding in all participants. This study is registered with EudraCT, number 2017-000908-21 and ClinicalTrials.gov, number NCT03430349. Between May 22 and Aug 22, 2017, 48 volunteers were screened, of whom 15 (31%) volunteers were excluded for reasons relating to the inclusion or exclusion criteria, three (6%) volunteers were not treated because of restrictions to the number of participants in each group, and 30 (63%) volunteers were sequentially allocated to groups (15 participants per group). Both novel OPV2 candidates were immunogenic and increased the median blood titre of serum neutralising antibodies; all participants were seroprotected after vaccination. Both candidates had acceptable tolerability, and no serious adverse events occurred during the study. However, severe events were reported in six (40%) participants receiving candidate 1 (eight events) and nine (60%) participants receiving candidate 2 (12 events); most of these events were increased blood creatinine phosphokinase but were not accompanied by clinical signs or symptoms. Vaccine virus was detected in the stools of 15 (100%) participants receiving vaccine candidate 1 and 13 (87%) participants receiving vaccine candidate 2. Vaccine poliovirus shedding stopped at a median of 23 days (IQR 15–36) after candidate 1 administration and 12 days (1–23) after candidate 2 administration. Total shedding, described by the estimated median shedding index (50% cell culture infective dose/g), was observed to be greater with candidate 1 than candidate 2 across all participants (2·8 [95% CI 1·8–3·5] vs 1·0 [0·7–1·6]). Reversion to neurovirulence, assessed as paralysis of transgenic mice, was low in isolates from those vaccinated with both candidates, and sequencing of shed virus indicated that there was no loss of attenuation in domain V of the 5ʹ-untranslated region, the primary site of reversion in Sabin OPV. We found that the novel OPV2 candidates were safe and immunogenic in IPV-immunised adults, and our data support the further development of these vaccines to potentially be used for maintaining global eradication of neurovirulent type-2 polioviruses. Bill & Melinda Gates Foundation.

Vaccination with Sabin, a live attenuated oral polio vaccine (OPV), results in robust intestinal and humoral immunity and has been key to controlling poliomyelitis. As with any RNA virus, OPV evolves rapidly to lose attenuating determinants critical to the reacquisition of virulence 1 – 3 resulting in vaccine-derived, virulent poliovirus variants. Circulation of these variants within underimmunized populations leads to further evolution of circulating, vaccine-derived poliovirus with higher transmission capacity, representing a significant risk of polio re-emergence. A new type 2 OPV (nOPV2), with promising clinical data on genetic stability and immunogenicity, recently received authorization from the World Health Organization for use in response to circulating, vaccine-derived poliovirus outbreaks. Here we report the development of two additional live attenuated vaccine candidates against type 1 and 3 polioviruses. The candidates were generated by replacing the capsid coding region of nOPV2 with that from Sabin 1 or 3. These chimeric viruses show growth phenotypes similar to nOPV2 and immunogenicity comparable to their parental Sabin strains, but are more attenuated. Our experiments in mice and deep sequencing analysis confirmed that the candidates remain attenuated and preserve all the documented nOPV2 characteristics concerning genetic stability following accelerated virus evolution. Importantly, these vaccine candidates are highly immunogenic in mice as monovalent and multivalent formulations and may contribute to poliovirus eradication. Mouse experiments and deep sequencing confirmed that two additional live attenuated vaccine candidates against type 1 and 3 polioviruses remained attenuated and preserved all documented nOPV2 characteristics concerning genetic stability following accelerated virus evolution.

Continued emergence and spread of circulating vaccine-derived type 2 polioviruses and vaccine-associated paralytic poliomyelitis from Sabin oral poliovirus vaccines (OPVs) has stimulated development of two novel type 2 OPV candidates (OPV2-c1 and OPV2-c2) designed to have similar immunogenicity, improved genetic stability, and less potential to reacquire neurovirulence. We aimed to assess safety and immunogenicity of the two novel OPV candidates compared with a monovalent Sabin OPV in children and infants. We did two single-centre, multi-site, partly-masked, randomised trials in healthy cohorts of children (aged 1–4 years) and infants (aged 18–22 weeks) in Panama: a control phase 4 study with monovalent Sabin OPV2 before global cessation of monovalent OPV2 use, and a phase 2 study with low and high doses of two novel OPV2 candidates. All participants received one OPV2 vaccination and subsets received two doses 28 days apart. Parents reported solicited and unsolicited adverse events. Type 2 poliovirus neutralising antibodies were measured at days 0, 7, 28, and 56, and stool viral shedding was assessed up to 28 days post-vaccination. Primary objectives were to assess safety in all participants and non-inferiority of novel OPV2 day 28 seroprotection versus monovalent OPV2 in infants (non-inferiority margin 10%). These studies were registered with ClinicalTrials.gov, NCT02521974 and NCT03554798. The control study took place between Oct 23, 2015, and April 29, 2016, and the subsequent phase 2 study between Sept 19, 2018, and Sept 30, 2019. 150 children (50 in the control study and 100 of 129 assessed for eligibility in the novel OPV2 study) and 684 infants (110 of 114 assessed for eligibility in the control study and 574 of 684 assessed for eligibility in the novel OPV2 study) were enrolled and received at least one study vaccination. Vaccinations were safe and well tolerated with no causally associated serious adverse events or important medical events in any group. Solicited and unsolicited adverse events were overwhelmingly mild or moderate irrespective of vaccine or dose. Nearly all children were seroprotected at baseline, indicating high baseline immunity. In children, the seroprotection rate 28 days after one dose was 100% for monovalent OPV2 and both novel OPV2 candidates. In infants at day 28, 91 (94% [95% CI 87–98]) of 97 were seroprotected after receiving monovalent OPV2, 134 (94% [88–97]) of 143 after high-dose novel OPV2-c1, 122 (93% [87–97]) of 131 after low-dose novel OPV2-c1, 138 (95% [90–98]) of 146 after high-dose novel OPV2-c2, and 115 (91% [84–95]) of 127 after low-dose novel OPV2-c2. Non-inferiority was shown for low-dose and high-dose novel OPV2-c1 and high-dose novel OPV2-c2 despite monovalent OPV2 recipients having higher baseline immunity. Both novel OPV2 candidates were safe, well tolerated, and immunogenic in children and infants. Novel OPV2 could be an important addition to our resources against poliovirus given the current epidemiological situation. Fighting Infectious Diseases in Emerging Countries and Bill & Melinda Gates Foundation.